Methods used to detect selected nucleotide sequences within target nucleic acids underlie an extensive array of practical applications including, but not limited to, paternity testing, forensic analysis, organ donor recipient matching, disease diagnosis, prognosis and treatment, and prenatal counseling.
There exists a need in the art for materials and methods that permit pluralities of selected nucleotide sequences to be simultaneously detected and analyzed, under uniform experimental conditions, preferably in a single, automated, assay reaction. One approach towards meeting this need has been the development of mobility-modifying polymers that can be attached to sequence-specific nucleobase polymers that act to increase the effective size of the modified nucleobase polymers. Where the charge to translational frictional drag ratio of the mobility-modifying polymer differs from that of the nucleobase polymer to which it is attached, the resulting modified nucleobase polymer will have an electrophoretic mobility that differs from that of the unmodified nucleobase polymer. This alteration of the charge to translational frictional drag ratio may be employed in various applications to effect electrophoretic separation of similarly-sized nucleobase polymers under both sieving and non-sieving conditions.
The most commonly employed mobility-modifying polymers are polyethylene oxides (PEO) that are attached to a nucleobase polymer using standard DNA chemistry (Grossman et al. (1994) Nucleic Acids Research 22 (21): 4527-34). An exemplary standard PEO phosphoramidite reagent (“PEO reagent”) that can be added to a nucleobase polymer using standard DNA chemistry is illustrated below:
In the illustration, DMT represents dimethoxytrityl and iPr represents isopropyl. When x=5, each PEO reagent added to the nucleobase polymer imparts the nucleobase polymer with an electrophoretic retardation of approximately 2 nucleotides as compared to the unmodified nucleobase polymer under both sieving and nonsieving electrophoretic conditions. Due to limitations of DNA chemistry, no more than about 40 PEO reagents can be coupled to a nucleobase polymer and result in homogenous product. Accordingly, the greatest electrophoretic mobility retardation that can be achieved using these standard PEO modifying reagents is about 80 nucleotides.
However, in light of the increasing need to simultaneously analyze vast numbers of nucleotide sequences in a single experiment, e.g., the 200 identified alleles associated with cystic fibrosis, there remains a need in the art for new mobility-modifying polymers that have different charge to translational frictional drag ratios than currently available mobility-modifying polymers, and that can impart electrophoretic mobility retardations of greater than the 80 nucleotides achievable with available PEO modifying reagents. The availability of such new mobility-modifying polymers would greatly increase the repertoire of available mobility modifications, thereby enabling the ability to perform extremely complex sequence analyses in simple, preferably automated, formats.